结直肠癌TP53相关外泌体的差异蛋白分析  

Analysis of differential proteins of TP53-related exosomes in colorectal cancer

作　　者：朱琳[1] 张泽 贾坤 周茜宁 周光[1] ZHU Lin;ZHANG Ze;JIA Kun;ZHOU Xining;ZHOU Guang(Department of Clinical Laboratory,the First Medical Center,Chinese PLA General Hospital,Beijing 100853,China;Department of Clinical Laboratory,the Sixth Medical Center,Chinese PLA General Hospital,Beijing 100037,China;Department of Clinical Laboratory,Beijing Friendship Hospital Affiliated to Capital Medical University,Beijing 100050,China)

机构地区：[1]解放军总医院第一医学中心检验科,北京100853 [2]解放军总医院第六医学中心检验科,北京100037 [3]首都医科大学附属北京友谊医院临床检验中心,北京100050

出　　处：《国际检验医学杂志》2022年第5期539-544,共6页International Journal of Laboratory Medicine

基　　金：军队青年成长课题(15QNP080)。

摘　　要：目的比较分析TP53突变、敲除及野生的结直肠癌细胞分泌外泌体的蛋白质组差异。方法从TP53野生型[HCT116-p53(WT)]、敲除型[HCT116-p53(-/-)]和构建的273位点突变型的结直肠癌HCT116细胞[HCT116-p53(R273H)]培养上清中分别提取外泌体,通过透射电镜观察其形态和免疫印迹法检测外泌体标志性蛋白,经同位素标记相对和绝对定量联合液相色谱串联质谱(iTRAQ-LC-MS/MS)策略分析3种细胞外泌体蛋白组成的差异,并通过酶联免疫吸附法(ELISA)进行差异蛋白验证。结果HCT116-p53(WT)分泌的外泌体和HCT116-p53(R273H)分泌的外泌体相比有144个差异蛋白,HCT116-p53(WT)分泌的外泌体和HCT116-p53(-/-)分泌的外泌体相比有480个差异蛋白,大部分差异蛋白主要参与组织代谢和细胞的生物学过程以及细胞的凋亡、应激、增殖、黏附等。通路分析(IPA)显示HCT116-p53(WT)分泌的外泌体相比于HCT116-p53(R273H)分泌的外泌体的差异蛋白主要富集在泛素化、mTOR、DNA损伤修复通路中;HCT116-p53(WT)分泌的外泌体相比于HCT116-p53(-/-)分泌的外泌体的差异蛋白主要集中在泛素化、EIF2、糖酵解通路中。结直肠癌患者的血清视网膜母细胞瘤蛋白(RB1)水平明显高于健康对照,差异有统计学意义(P=0.0012)。结论TP53突变和缺失会改变外泌体的蛋白组成,富集许多泛素化蛋白,结直肠癌患者的血清RB1水平明显高于健康对照组,这为深入探讨TP53状态对外泌体蛋白组成和肿瘤微环境的影响提供依据。Objective To compare and analyze the proteome differences of TP53 mutation,knockout and wild-type colorectal cancer cells secreting exosomes,and to explore the effect of mutant TP53 on secreted proteins in the tumor microenvironment.Methods Separate exosomes from culture supernatant of TP53 wild-type HCT116-p53(WT),knockout type HCT116-p53(-/-)and constructed 273 site mutant colorectal cancer cells HCT116-p53(R273H).Observe its morphology by transmission electron microscope and detect exosomal marker protein by Western blotting.The difference in protein composition of the three exosomes was analyzed by the iTRAQ-LC-MS/MS strategy,and the differential protein was verified by enzyme-linked immunosorbent assay(ELISA).Results There were 144 differential proteins between HCT116-p53(WT)secreted exosomes and HCT116-p53(R273h)secreted exosomes,and 480 differential proteins between HCT116-p53(WT)secreted exosomes and HCT116-p53(-/-)secreted exosomes.Most of the differential proteins were mainly involved in metabolism and cell biological processes,as well as apoptosis,stress,proliferation,cell adhesion and other functions.Pathway analysis(IPA)showed that the differential proteins secreted by HCT116-p53(WT)were mainly enriched in ubiquitination,mTOR and DNA damage repair pathways compared with those secreted by HCT116-p53(R273H).Compared with the secretion secreted by HCT116-p53(-/-),the differential proteins secreted by HCT116-p53(WT)were mainly concentrated in ubiquitination,EIF2 and glycolysis pathways.Moreover,the level of serum retinoblastoma protein(RB1)in patients with colon cancer was significantly higher than that in healthy controls,the difference was statistically significant(P=0.0012).Conclusion Mutations and deletions of TP53 will change the protein composition of exosomes,enriching many ubiquitinated proteins.The serum RB1 level of colorectal cancer patients was significantly higher than that of healthy controls.This provides a basis for exploring the influence of TP53 status on exosomal protein composition an

关 键 词：外泌体 TP53基因 结直肠癌 质谱分析 

分 类 号：R730.2[医药卫生—肿瘤]